Methods and apparatus for gas stream mass transfer with a liquid

ABSTRACT

A system for performing a gas-liquid mass transfer includes a container bounding a compartment and having a top wall, a bottom wall, and an encircling sidewall extending therebetween. A tube has a first end and an opposing second end, the first end of the tube being disposed within the compartment of the container. A nozzle is disposed within the compartment of the container and has at least one outlet, the nozzle being coupled with the tube so that a gas can be passed through the tube and out the at least one outlet of the nozzle. The nozzle is sufficiently buoyant so that when a fluid is disposed within the compartment of the container, the nozzle floats on the fluid.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/916,635, filed Mar. 9, 2018, which is a divisional of U.S.application Ser. No. 15/187,258, filed Jun. 20, 2016, now U.S. Pat. No.9,932,553, which is a divisional of U.S. application Ser. No.14/395,728, filed Oct. 20, 2014, now U.S. Pat. No. 9,388,375, which is aUS Nationalization of PCT/US2013/032528, filed Mar. 15, 2013, whichclaims priority to U.S. Provisional Application No. 61/625,794, filedApr. 18, 2012, which applications are incorporated herein by specificreference.

BACKGROUND OF THE INVENTION 1. The Field of the Invention

The present invention relates to methods and systems for producing agas-liquid mass transfer which, in one example, can be used foroxygenating biological cultures having a shallow depth within a reactor.

2. The Relevant Technology

The growth of biological cells within a bioreactor requires criticalcontrol over a number of different process parameters. For example, ascells grow, they absorb oxygen from the surrounding media and releaseCO₂. The concentration of oxygen and CO₂ within the media must becarefully monitored and regulated to ensure viability and optimal growthof the cells. Another factor that needs to be carefully monitored andcontrolled is the density of the cells within the culture. To make surethat all of the processing parameters are properly controlled, cells aretypically grown in sequential stages of increasingly larger reactors.For example, cell cultures may initially start in a small flask. Oncethe cell density approaches a critical value, the culture is transferredto a larger bench top reactor where the culture is combined withadditional media. In turn, once the cell density again reaches acritical value, the culture is again moved to a larger reactor with moremedia. This process continues until a desired volume of culture isachieved. Because each different sized reactor only processes theculture over a relatively narrow change in volume, conventionaltechniques can be used for controlling all of the process parameters.

Although the above method of production works, there are a number ofdisadvantages in having to transfer the cell culture to differentcontainers during the growth process. For example, the process is timeconsuming, labor intensive, and requires that the producer obtain andmaintain a relatively large number of different sized reactors. Inaddition, the process of transferring the culture temporarily halts thepreferred processing conditions, can potentially damage the cells, andincreases the risk of a breach in sterility. Attempts have been made toovercome some of the above problems by trying to process a large changein volume of culture within a single reactor. For example, in contrastto conventional reactors which may only see a change in the volume ofculture by a factor of two, attempts have been made to increase thechange in the volume of a culture within a reactor by a factor of five.

The concept is to start with a small volume of culture within arelatively large reactor container and then through batch or continuousfeed mode continue to add media to the culture as the cells grow to apoint where the container reaches a predefined maximum volume ofculture. Depending upon how much culture is needed, the culture canstill be transferred to a larger reactor. The goal is to reduce thenumber of different reactors/containers the culture needs to betransferred into before reaching the desired end volume.

There are, however, a number of complications in growing a culturewithin a single reactor over a large change in volume. For example, ineach reactor there is a mechanism for oxygenating the culture, strippingout unwanted CO₂, and continuously mixing the culture so that theculture remains substantially homogeneous. Mixing is commonlyaccomplished by an impeller disposed within the container. The impelleris sized, positioned and operated so as to achieve optimal mixing of theculture without damaging the cells. Oxygenation is typicallyaccomplished by dispersing small diameter bubbles into the containerholding the culture through a defined sparger located on the floor ofthe container. As the bubbles rise within the culture, the oxygen isabsorbed into the culture. CO₂ stripping is typically accomplished bydispersing large diameter bubbles into the container through a secondsparger located on the floor of the container. As the large bubbles risewithin the culture, a portion of the CO₂ within the culture equilibratesinto the air of the large bubbles and is carried out of the culture.

One of the complications of growing a culture within a single reactorover a large change in volume is that the parameters for oxygenating,stripping CO₂ and mixing a culture, along with other operatingparameters, change as the volume of culture increases. Traditionalmechanisms, as discussed above, for oxygenating, stripping CO₂ andmixing are designed to operate over a narrow range of fluid volumes andthus for a set configuration size do not effectively function at bothsmall and large fluid volumes. The same is also true when other gases,such as nitrogen, are desired to be applied to the culture. Accordingly,what is needed in the art are methods and systems for oxygenating aculture and/or stripping CO₂ from a culture and, more generically,creating a gas-liquid mass transfer with a culture that solves all orsome of the above problems and can effectively operate in conditionswhere traditional sparger mechanisms have difficulty performingcorrectly.

BRIEF DESCRIPTION OF THE DRAWINGS

Various embodiments of the present invention will now be discussed withreference to the appended drawings. It is appreciated that thesedrawings depict only typical embodiments of the invention and aretherefore not to be considered limiting of its scope.

FIG. 1 is a cross sectional side view of a reactor system containing aculture;

FIG. 2 is a partial cross sectional side view of one embodiment of a gasdelivery system shown in FIG. 1;

FIG. 3 is a partial cross sectional side view of an alternativeembodiment of a gas delivery system shown in FIG. 1;

FIG. 4 is a partial cross sectional side view of another alternativeembodiment of a gas delivery system in a first position that can be usedwith the reactor shown in FIG. 1;

FIG. 5 is a partial cross sectional side view of the gas delivery systemshown in FIG. 4 in a second position;

FIG. 6 is a partial cross sectional side view of an alternativeembodiment of the gas delivery system shown in FIG. 4;

FIG. 7 is a cross sectional side view of the reactor system shown inFIG. 1 including a further alternative embodiment of a gas deliverysystem that adjustably extends down from the upper end wall of thecontainer;

FIG. 8 is a cross sectional side view of the reactor system shown inFIG. 1 that includes a further alternative embodiment of a gas deliverysystem comprising a multi-lumen tube having a plurality of spaced apartnozzles connected thereto;

FIG. 9 is a cross sectional side view of the tube shown in FIG. 8 takenalong section lines 9-9 in FIG. 8; and

FIG. 10 is a cross sectional side view of the nozzle shown in FIG. 8taken along section lines 10-10 in FIG. 8.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

As used in the specification and appended claims, directional terms,such as “top,” “bottom,” “left,” “right,” “up,” “down,” “upper,”“lower,” “proximal,” “distal” and the like are used herein solely toindicate relative directions and are not otherwise intended to limit thescope of the invention or claims.

The present invention relates to novel methods and systems forefficiently producing a gas-liquid mass transfer and for particularlyproducing a gas-liquid mass transfer with shallow volumes of liquid. Inone embodiment, the methods and systems can be used in oxygenating abiological culture disposed within a reactor container and particularlycultures having a relatively shallow depth. For example, the methods andsystems can commonly be used in bioreactors and fermentors for culturingcells or microorganisms. Specifically, the inventive methods and systemscan be used in culturing bacteria, fungi, algae, plant cells, animalcells, protozoan, nematodes, and the like. The methods and systems canalso be used in association with the formation and/or treatment ofsolutions and/or suspensions that are for biological purposes, such asmedia, buffers, or reagents. For example, the methods and systems can beused in the formation of media where sparging is used to control the pHof the media through adjustment of the carbonate/bicarbonate levels withcontrolled gaseous levels of carbon dioxide. In other applications, themethods and systems can be used for stripping gases, such as oxygen orCO₂ from a culture or fluid. It is appreciated that the inventivemethods and systems are not limited to being used with biologicalcultures but can also be used in food production, chemical production,biopharmaceutical production and other types of production where agas-liquid mass transfer is desired.

In general, one embodiment of the inventive method comprises passing astream of a gas over a top surface of a liquid at a sufficient velocityand direction so that the gas stream produces turbulence on the topsurface of the liquid that is sufficient to produce a mass transferbetween the stream of gas and the liquid. This process is referred toherein as “gas stream mass transfer” or, where the process is used foroxygenating a fluid, the process can be referred to as “gas streamoxygenation.” The process is similar to how wind passing over thesurface of a lake creates Langmuir circulations to oxygenate the lakewater. That is, as a result of the gas stream flowing over the surfaceof the liquid, there is both an efficient mass transfer of the gas intothe fluid and there is a vertical circulation of the fluid near thesurface. This circulation of the fluid ensures that the upper layer ofthe fluid has a uniform gas concentration. In turn, an impeller or othermixing system can be used to ensure that the upper layer of the fluid isuniformly mixed throughout the remainder of the fluid so that the entirefluid has a proper gas concentration. In other applications, asmentioned above and as will be discussed below in greater detail, thesame process of passing a gas stream over the top surface of a fluid soas to produce fluid turbulence can be used for stripping gas out of thefluid.

Although gas stream mass transfer is primarily discussed herein withregard to oxygenating a biological culture, the same methods and systemscan also be used for oxygenating other types of liquids, such as thosementioned above. In addition, as discussed below in greater detail, theinventive methods and systems are not limited to oxygenating a fluid butcan be used with other gases for affecting any type of mass transferinto a liquid and/or out of a liquid.

Gas stream mass transfer has a number of processing advantages when itis used for oxygenating a biological culture within a reactor container,particularly over conventional sparging techniques. Where a reactorcontainer is being designed to process a culture of cells ormicroorganisms over a relatively large change in fluid volume, thediameter of the container typically needs to be relatively large tomaintain geometry and height requirements. As the diameter of thecontainer increases with respect to volume, the depth of the culturewithin the container decreases. As a result, for very small volumes ofculture within the container, such as when the initial volume of cultureis transferred into the container, the resident time for the oxygenatingbubbles that are typically sparged into the culture from the floor ofthe container is insufficient to properly oxygenate the culture. Thatis, because the depth of culture is so shallow, the oxygenating bubblesare not within the culture for a sufficient period of time to fullyoxygenate the culture as the bubbles travel from the sparger to the topsurface of culture. Likewise, the resident time for the larger spargedbubbles used to strip out the CO₂ is also insufficient to fully removethe unwanted CO₂ from the culture. This problem is further compounded bythe fact that the CO₂ gas is heavier than air so that the CO₂ lays likea blanket over the top surface of the culture, thereby further hamperingoxygenation of the culture and removing CO₂.

In contrast to sparging which becomes more efficient as the depth of theculture increases, gas stream oxygenation or mass transfer, which isaccomplished by blowing a stream of air or other gas containing oxygenover the top surface of the culture, become more efficient as the depthof the culture or other fluid being processed decreases. Thus, gasstream oxygenation is particularly useful for shallow depth culturesdisposed within a reactor; including reactors that start with a smallvolume and increase to a large volume. In addition, sparging is known toproduce unwanted foam on the top surface of cultures, especially whenthe spargers used generate very small bubbles (sub millimeter diameter).In contrast, gas stream mass transfer produces minimal foaming and canassist in reducing the vessel foam generation by reducing the amount oftraditional sparging required. Furthermore, gas stream oxygenationprevents the formation of a CO₂ blanket on the surface of the culture.As such, the gas on the surface of the culture is both well controlledand well mixed, permitting the CO₂ to dissipate out of the culture, mixinto the head space of the reactor, and leave via the system exhaustport. The interaction of the gas stream oxygenation with the systemliquid also helps directly facilitate stripping CO₂ from the culture.Accordingly, for relatively shallow depth cultures, gas streamoxygenation can be used to both oxygenate the culture and remove CO₂from the culture, in some cases eliminating the need for traditionalsparging in certain forms of the invention.

As the depth of a culture within a reactor increases, the efficiency ofoxygenating the culture at the bottom of the reactor through gas streamoxygenation decreases. Accordingly, as the depth of the cultureincreases, dO₂ sensors or other parameters or mechanisms can be used todetermine when sparging or other methods of oxygenation should beactivated. That is, as the depth of the culture increases, sparging canbe activated such as through stepped increments or through continuousgradual increase so as to ensure that the culture is always properlyoxygenated. The applied gas stream oxygenation can decrease as spargingincreases or can remain constant. Even if the gas stream is not fullyoxygenating the culture, the gas stream is still equilibrating the upperregion of the culture and preventing CO₂ blanketing which in turnassists in traditional sparge operation. Thus, even for relatively deepvolumes of culture, gas stream oxygenation can continue to be used inconjunction with sparging or other methods of oxygenation. It should beappreciated that an electronic controller could be used to automaticallyactivate and/or regulate sparging and gas flow based on sensor readings.

Turning to the Figures, examples of systems will now be discussed thatcan be used in performing gas stream oxygenation/mass transfer. Depictedin FIG. 1 is one embodiment of a reactor system 10 incorporatingfeatures of the present invention. In general, reactor system 10comprises a support housing 12 that bounds a chamber 14, a containerassembly 16 disposed within chamber 14 and a mixing system 17 coupledwith container assembly 16. Support housing 12 typically comprises arigid tank, such as a metal tank. The tank can be jacketed forcontrolling the temperature of the culture within container assembly 16.Support housing 12 can be any desired size, shape, or configuration thatwill properly support container assembly 16, as discussed below.

With continued reference to FIG. 1, container assembly 16 comprises acontainer 18 having a side 20 that extends from an upper end 22 to anopposing lower end 24. Upper end 22 terminates at an upper end wall 33while lower end 24 terminates at a lower end wall 34. Container 18 alsohas an interior surface 26 that bounds a compartment 28. Compartment 28is configured to hold a fluid. The fluid can comprise a biologicalculture which comprises cells or microorganisms, media, and othernutrients and additives. Any other type of fluid can also be used thatrequires mass transfer with a gas. For example, the fluid can be achemical, biological fluid, food product, or other fluid. For theexample herein, the fluid will be discussed as biological culture 29.Culture 29 has a top surface 31. A head space 37 is disposed withincompartment 28 and is bounded between top surface 31 of culture 29 andupper end wall 33.

In the embodiment depicted, container 18 comprises a flexible bag thatis comprised of a flexible, water impermeable material such as alow-density polyethylene or other polymeric sheets or film having athickness in a range between about 0.1 mm to about 5 mm with about 0.2mm to about 2 mm being more common. Other thicknesses can also be used.The material can be comprised of a single ply material or can comprisetwo or more layers which are either sealed together or separated to forma double wall container. Where the layers are sealed together, thematerial can comprise a laminated or extruded material. The laminatedmaterial comprises two or more separately formed layers that aresubsequently secured together by an adhesive. Examples of extrudedmaterial that can be used in the present invention include the HyQ CX3-9and HyQ CX5-14 films available from HyClone Laboratories, Inc. out ofLogan, Utah. The material can be approved for direct contact with livingcells and be capable of maintaining a solution sterile. In such anembodiment, the material can also be sterilizable such as by ionizingradiation. Prior to use, container assembly 16 is typically sealedclosed and sterilized so that compartment 28 is sterile prior to theintroduction of culture 29.

In one embodiment, container 18 can comprise a two-dimensional pillowstyle bag. In another embodiment, container 18 can be formed from acontinuous tubular extrusion of polymeric material that is cut tolength. The ends can be seamed closed or panels can be sealed over theopen ends to form a three-dimensional bag. Three-dimensional bags notonly have an annular sidewall but also a two dimensional top end walland a two dimensional bottom end wall. Three dimensional containers cancomprise a plurality of discrete panels, typically three or more, andmore commonly four or six. Each panel is substantially identical andcomprises a portion of the sidewall, top end wall, and bottom end wallof the container. Corresponding perimeter edges of each panel are seamedtogether. The seams are typically formed using methods known in the artsuch as heat energies, RF energies, sonics, or other sealing energies.

In alternative embodiments, the panels can be formed in a variety ofdifferent patterns. Further disclosure with regard to one method ofmanufacturing three-dimensional bags is disclosed in US Publication No.US 2002-0131654 A1, published Sep. 19, 2002, which is incorporatedherein by specific reference in its entirety.

It is appreciated that container 18 can be manufactured to havevirtually any desired size, shape, and configuration. For example,container 18 can be formed having a compartment sized to 10 liters, 30liters, 100 liters, 250 liters, 500 liters, 750 liters, 1,000 liters,1,500 liters, 3,000 liters, 5,000 liters, 10,000 liters or other desiredvolumes. The size of the compartment can also be in the range betweenany two of the above volumes. Although container 18 can be any shape, inone embodiment container 18 is specifically configured to be generallycomplementary to chamber 14 of support housing 12 in which container 18is received so that container 18 is properly supported within chamber14.

Although in the above discussed embodiment container 18 is depicted as aflexible bag, in alternative embodiments it is appreciated thatcontainer 18 can comprise any form of collapsible container orsemi-rigid container. In still other embodiments, container 18 can berigid and support housing 12 can be eliminated.

Continuing with FIG. 1, formed on container 18 are examples of aplurality of different ports that can be mounted thereon with each ofthe ports communicating with compartment 28. Specifically, mounted onupper end wall 33 are access ports 40 and 41 having lines 39A and Bcoupled therewith, respectively. Access ports 40 and 41 can be used fordelivering gas, media, cultures, nutrients, and/or other components intocontainer 18 and can be used for withdrawing culture 29 or gas fromwithin head space 37. For example, in some forms of the invention, port40 can be used as a gas inlet into head space 37 and port 41 can be usedas a gas outlet from head space 37. Any desired number of access portscan be formed on container 18. A sensor port 42 is formed on side 20 ofcontainer 18. A sensor 50 is disposed within sensor port 42 so as tocommunicate with compartment 28, typically at the lower end thereof. Itis appreciated that any number of sensor ports 42 can be formed oncontainer 18 each having a corresponding sensor 50 disposed therein.Examples of sensors 50 that can be used include temperatures probes, pHprobes, dissolved oxygen sensors, carbon dioxide sensors, cell masssensors, nutrient sensors, and any other sensors that allow for testingor checking the culture or production. The sensors can also be in theform of optical sensors and other types of sensors.

Mounted on lower end wall 34 are sparging ports 43 and 44. A firstsparger 52 is mounted to port 43 and is designed to deliver smallbubbles to culture 29 for oxygenating culture 29. Sparger 52 can beformed integral with or attached to port 43. A second sparger 54 ismounted to port 44 and is designed to deliver larger bubbles to culture29 for stripping CO₂ from culture 29. As such, the bubbles from firstsparger 52 are smaller than the bubbles from second sparger 54. In someforms of the invention, second sparger 54 can be an open tube or a tubewith a porous frit with relatively large pores, while first sparger 52can be a tube with a porous frit with relatively small pores. Firstsparger 52 can also comprise a perforated or porous membrane that ismounted on the end of port 43 or on the interior surface of lower endwall 34 so as to extend over port 43. It is appreciated that spargerscome in a variety of different configurations and that any type ofspargers can be used as desired or as appropriate for the expectedculture volumes, cells and conditions.

It is again noted that container 18 can be formed with any desirednumber of ports and that the ports can be formed at any desired locationon container 18. The ports can be the same configuration or differentconfigurations and can be used for a variety of different purposes suchas listed above but not limited thereto. Examples of ports and howvarious probes, sensors, and lines can be coupled thereto is disclosedin US Publication No. 2006-0270036, published Nov. 30, 2006 and USPublication No. 2006-0240546, published Oct. 26, 2006, which areincorporated herein by specific reference in their entirety. The portscan also be used for coupling container 18 to secondary containers, tocondenser systems, and to other desired fittings.

Also disposed along side 20 of container 18 are a plurality ofvertically spaced apart gas ports 45-47. Each of ports 45-47 forms partof a corresponding gas delivery system, which systems are designed fordelivering gas into compartment 28 to produce gas streamoxygenation/mass transfer. Depicted in FIG. 2 is an enlarged view of agas delivery system 60A which includes gas port 45. Port 45 comprises aflange 62 mounted to container 18 and a tubular stem 64 outwardlyprojecting therefrom. Stem 64 bounds a passageway 66 longitudinallyextending therethrough so as to communicate with compartment 28. Anannular barb 68 is formed on the free end of stem 64 and couples with atube 70. In turn, tube 70 couples with an aseptic connector 72.

Aseptic connector 72 includes a first connector portion 74 thatselectively mates with and fluid couples to a second connector portion76. A tubular stem 75 projects from first connector portion 74 and fluidcouples with tube 70. Each of connector portions 74 and 76 have asealing layer 78A and B, respectively, that covers the opening toconnector portions 74 and 76. After connector portions 74 and 76 arecoupled together, sealing layers 78A and B are pulled out from betweenthe connector portions so as to form an aseptic fluid connection betweenconnector portions 74 and 76. Aseptic connectors are known in the art.One example of an aseptic connector is the KLEENPACK® connector producedby the Pall Corporation. The PALL connector is described in detail inU.S. Pat. No. 6,655,655, the content of which is incorporated herein byreference in its entirety. Other aseptic connectors can also be used.

A tube 80 fluid couples with second connector portion 76 and extends toa gas supply 82. Gas supply 82 delivers a gas which passes throughaseptic connector 72, port 45 into compartment 28. The gas can be oxygenor it can be a gas containing oxygen, such as air. Other gases can alsobe used depending on the desired application. Gas supply 82 can comprisea pressurized canister, a compressor, or other gas supply source.Disposed along tube 80 is a gas filter 84 that sterilizes the gas as itpasses therethrough. Also mounted along tube 80 is a valve 86. Valve 86is used to selectively stop the flow of gas through delivery system 60Aand to prevent culture 29 within container 18 from flowing out throughdelivery system 60. Valve 86 can have a variety of differentconfigurations. For example, valve 86 can comprise a ball valve, a gatevalve, a clamp that pinches tube 80 or any other type of valve thatfunctions for the intended purpose. Valve 86 can be manually controlledor can be electric, hydraulic, pneumatic or the like. It is appreciatedthat valve 86 can be positioned anywhere along delivery system 60 but istypically located close to gas port 45. In one embodiment, valve 86 canbe mounted on tube 70 adjacent to port 45 or directly on port 45.

As previously discussed, the object of gas delivery system 60 is todeliver a stream of gas over top surface 31 of culture 29 or otherapplicable fluid at a sufficient velocity and direction so that the gasstream produces a turbulence on top surface 31 that is sufficient tooxygenate the culture for growing the cells or microorganisms therein.The term “over” is broadly intended to include the gas traveling overtop surface 31 in any desired orientation such as horizontal,substantially horizontal, downwardly inclined, or upwardly inclined. Thegas stream need not flow in a linear path but can flow in a circularpath or vortex, such as about a vertical or horizontal axis, or can flowalong a random path. The gas stream can be a laminar flow or a turbulentflow and the direction, flow rate, and/or speed of the gas flow can beconstant or variable. For example, the gas stream can change from adownward vertical direction to a substantially horizontal direction. Byplacing gas port 64 on side 20 of container 18, the gas passing outthrough passageway 66 in this embodiment travels horizontally orsubstantially horizontally within compartment 28 so that it can passover and across top surface 31. In some embodiments, the gas streamoxygenation can be sufficient to independently oxygenate the culture tothe extent needed for growing the cells or microorganisms without anyother form of oxygenation, such as sparging. In other embodiments, thegas steam oxygenation can be used in conjunction with sparging or otheroxygenation processes.

In one embodiment, the gas stream oxygenation is able to achieve a masstransfer of oxygen using only air and without the aid of sparging havinga kLa factor that is greater than 3 and more commonly greater than 5 or7. The gas stream oxygenation can also maintain, without separatesparging, a stable oxygen concentration set point within the activeculture that is in a range of 30%-50% of air saturation. The abovevalues can be achieved in a stirred tank reactor with mixing by impellerand in other types of rectors. In one specific example, gas streamoxygenation, using only air, was able to oxygenate a CHO culture at atarget value of 50% of air saturation (868 mbar ambient pressure) andstrip CO₂ to a cell concentration of 3.5E+06 cell/mL at ⅕^(th) vesselvolume. At this point the culture was then fed media to full vesselvolume. It is worth noting that the oxygenation and CO₂ strippingprovided by the gas stream oxygenation was excessive at this level ofculture density and vessel fill volume; it required the addition of N₂and CO₂ mixed in with the air to hold target pH and dissolved O₂ targetvalues.

During operation, compartment 28 of container 18 is filled with culture29 so that top surface 31 is disposed close to passageway 66. In oneembodiment, the distance D₁ between passageway 66 and top surface 31 isin a range between about 0.75 cm to about 15 cm with about 1 cm to about10 cm or about 2 cm to about 5 cm being more common. Other distances canalso be used. Furthermore, the distance D₁ can vary based upon factorssuch as the size of container 18, the projection angle of the gas (withflow perpendicular to the liquid surface being optimal), the flow rateof the gas, and the superficial velocity of the gas. When measuring thedistance D₁, top surface 31 can be the maximum liquid wave height underagitation of culture 29 or can be top surface 31 with no agitation. Forscalable representation, the flow rate can be measured in rate of VesselVolumes per Minute (VVM) of the maximum rated liquid working volume ofthe system. The flow rate of the gas passing out through passageway 66is typically in a range between about 0.06 VVM to about 0.2 VVM withabout 0.08 VVM to about 0.1 VVM or about 0.16 VVM to about 0.18 VVMbeing more common. Other flow rates can also be used depending on theintended application. The velocity of the gas exiting passageway 66 ortraveling across top surface 31 within compartment 28 is typically in arange between about 25 m/sec to about 275 m/sec with about 25 m/sec toabout 175 m/sec or about 30 m/sec to about 100 m/sec being more common.The velocity can be greater than 25 m/sec and more commonly greater than40 m/sec, 60 m/sec, 80 m/sec, or 100 m/sec. To achieve desired gasvelocities exiting passageway 66, passageway 66 can have a minimum exitarea of flux based on the volume of compartment 12, i.e., vessel volume(VV). This minimum exit area of flux can be in a range between about VV(liters)/80 (liters/mm²) to about VV (liters)/7.8 (liters/mm²) withabout VV (liters)/40 (liters/mm²) to about VV (liters)/30 (liters/mm²)or about VV (liters)/8.5 (liters/mm²) to about VV (liters)/6.25(liters/mm²) being more common. Other areas can also be used.

If desired, port 45 can be configured so that during operation stem 64is angled so that the gas passing out therethrough is directed slightlydown towards top surface 31. For example, stem 64 has a centrallongitudinal axis 88. Port 45 can be formed so that axis 88 of stem 64is tilted relative to horizontal during use by an angle at in a rangebetween 1° to about 10° so that the gas passing out therethrough passesslightly down against top surface 31. Other angles can also be used.

As previously discussed, gas stream oxygenation is most efficient forshallow depths of culture 29 within container 18. In one embodiment, themaximum distance D₂ (FIG. 1) between top surface 31 and lower end wall34 at which the gas stream oxygenation can independently oxygenateculture 29 to grow cells or microorganisms can be in a range ofdistances based on diameter of the container 18, i.e., vessel diameter(VD). For example, maximum distance D₂ can be in a range between aboutVD (cm)*0.3 to about VD (cm)*0.4. Where container 18 does not have acircular transverse cross section, VD can be based on an averagediameter. In some specific examples, D₂ can be in a range between about5 cm to 30 cm or between 10 cm and 100 cm depending on the diameter ofthe container. Other distances can also be used. At some depths, thesystem can operate without the use of sparging or other oxygenationsystems. In addition, for some depths desired oxygenation can beachieved throughout the culture without the use of a separate mixer dueto the natural circulation caused by the blowing gas. As the depthincreases, however, proper oxygenation of the culture requires both gassteam oxygenation and a separate mixing system, such as thorough animpeller or rocking, to ensure all of the culture is properlyoxygenated.

As the depth of culture 29 increases, sensors 50 may detect the need foradditional oxygenation, even when mixing is being accomplished. Anelectrical controller or manual regulator can then be used to regulatethe flow of sparged gas through spargers 52 and 54 for furthercontrolling the oxygenation and CO₂ levels within culture 29. Althoughsparging with air or oxygen may not be required at shallow depths whenusing gas steam oxygenation, sparging with nitrogen, such as throughsparger 54, may still be used at all depths to control the oxygen withinthe culture, i.e., to strip out excess oxygen produced by gas steamoxygenation. Although gas delivery system 60A is shown in FIG. 1 as theonly gas delivery system that is located at or near the elevation oncontainer 18 corresponding to the top of distance D₂, two or more gasdelivery systems 60A can be located and simultaneously operated at ornear that same elevation.

The gas delivered to container 18 through gas delivery system 60A can bedrawn out through access port 41 so that container 18 does not overinflate. Because of the rather high volume of gas passing throughcontainer 18, there can be a higher rate of evaporation of the mediarelative to conventional systems. As such, reactor system 10 can beoperated with a condenser that couples with access port 41. One exampleof a condenser that can be used with reactor system 10 is disclosed inUS Publication No. 2011/0207218 A1, published Aug. 25, 2011, which isincorporated herein by specific reference in its entirety.

Culture 29 continues to grow at a level below passage 66 until a definedmass density or other desired value is determined within culture 29.Valve 86 can then be closed and media and other components added toculture 29 until the level of top surface 31 is raised to within anoperating distance from a second gas delivery system 60B shown inFIG. 1. Gas delivery system 60B is then activated to again pass a gasstream over top surface 31 and thereby continue with the gas streamoxygenation of culture 29. This process can then be continued for asubsequent gas delivery system 60C. Likewise, any number of additionalgas delivery systems can be vertically spaced apart along side 20 ofcontainer 18 for continuing gas stream oxygenation at other elevations.

In one embodiment, each of gas delivery systems 60A-C can be coupled toa separate gas supply 82 (FIG. 2). In an alternative embodiment,however, as depicted in FIG. 7, a manifold 161 can be fluid coupled toeach gas delivery system 60A-C while a single gas supply 82 is coupledwith manifold 161. Valves 86A-C incorporated into gas delivery systems60A-C, respectively, can be opened and closed by a common controller toregulate which gas delivery system 60A-C is opened for gas to traveltherethrough.

It is appreciated that each of gas delivery systems 60A-C can have thesame configuration as gas delivery system 60A. In alternativeembodiments, gas delivery systems 60A-C can have a differentconfiguration or the gas delivery systems can be different from eachother. For example, depicted in FIG. 3 is an enlarged view of a gasdelivery system 60B. Like elements between gas delivery systems 60A and60B are identified by like reference characters. In contrast to gasdelivery system 60A which uses port 45 having a rigid barbed stem 64,gas delivery system 60B uses gas port 46 having a flange 90 secured tocontainer 18 and a tubular stem 92 outwardly projecting therefrom.Flange 90 and stem 92 are comprised of a resiliently flexible materialtypically having a durometer on a Shore A scale with a value of lessthan 90. Further disclosure with regard to port 46 is disclosed in USPublication No. 2006-0240546, which was previously incorporated hereinby specific reference.

Stem 92 bounds a passageway 94 that communicates with compartment 28.Disposed within passageway 94 is a nozzle 96 that is secured to stem 92by a pull tie 97 or other type of clamp. An annular lip seal 99 inwardlyprojecting from stem 92 can from a liquid tight seal about nozzle 96.Nozzle 96 is tubular having an encircling sidewall 98 that bounds apassageway 100 extending therethrough. Nozzle 96 has a tip 102 whichbounds an outlet 104 through which the gas passes from passageway 100into compartment 28. Nozzle 96 is configured so that outlet 104 has thedesired size and configuration to achieve the desired gas velocity andflow rate to achieve gas stream oxygenation. The distances, dimensions,velocities, flow rates, orientations and the like discussed above withregard to gas delivery system 60A and passageway 66 are also applicableto gas delivery system 60B and passageway 100/outlet 104. Althoughnozzle 96 is shown having a single outlet 104 formed thereon, inalternative embodiments, nozzle 96 can be formed with a plurality ofradially spaced apart outlets 104 so that the gas stream fans out acrossmore of the surface of top surface 31. As a result of using nozzle 96, astandardized port 46 can be used on container 18 while a specificallydesigned nozzle 96 can be used for achieving the desired gas flowconditions.

Depicted in FIG. 4 is another alternative embodiment of a gas deliverysystem 110 for delivering a gas stream to compartment 28 of container18. Again, like elements with prior embodiments are identified by likereference characters. Gas delivery system 110 comprises port 46 havingflange 60 coupled to container 18 and tubular stem 92 outwardlyprojecting therefrom. Fluid coupled to the end of stem 92 is firstconnector portion 74 of aseptic connector 72 which can be selectivelycoupled with second connector portion 76. Coupled with the upstream sideof second connector portion 76 is a tubular sleeve 118. Tubular sleeve118 has a first end 120 coupled with second connector portion 76 and anopposing second end 122. Sleeve 118 is comprised of a flexible materialthat can be easily collapsed, such as by folding, gathering, compressingor the like, along its length. For example, sleeve 118 can be comprisedof a polymeric sheet or film. As such, tubular sleeve 118 can be in theform of a flexible bag having openings at opposing ends. In analternative embodiment, sleeve 118 can comprise a molded tube whereinthe encircling wall is accordioned for easy collapsing and expansion.Other configurations can also be used so that sleeve 118 folds, gathers,or compresses as second end 122 is pushed towards first end 120.

Tubular sleeve 118 bounds a chamber 124. Disposed within chamber 124 isan end portion of a flexible tube 126. Tube 126 has a first end 128disposed within chamber 124 and an opposing second end 130. Disposed onfirst end 128 of tube 126 is a nozzle 132 having an outlet 134 formedthereon. A clamp 136 encircles sleeve 118 at second end 122 andcompresses against the exterior surface of tube 126 so as to securesleeve 118 and tube 126 together and form a liquid tight sealtherebetween. As will be discussed below in greater detail, the portionof tube 126 within sleeve 118 can be formed having a resilient curvedarch along the length thereof. Second end 130 of tube 126 eitherdirectly or indirectly couples with gas supply 82 and can have gasfilter 84 and valve 86 disposed there along (FIG. 2).

During operation, gas delivery system 110 can be operated in a number ofdifferent positions. For example, once connector portions 74 and 76 arecoupled together and sealing layers 78 removed, an opening is formedthrough aseptic connector 72 that communicates with chamber 124 ofsleeve 118. In this configuration, second end 122 of sleeve 118 can bemanually pushed towards first end 122. Because tube 126 is secured tosleeve 118 by clamp 136, the advancing of second end 122 causes firstend 128 of tube 126 to concurrently advance through aseptic connector 72and into or through passageway 94 of port 46. Where top surface 31 ofculture 29 is disposed adjacently below passageway 94 of port 46, nozzle132 can remain disposed with passageway 94 or can extend slightly intochamber 28 for blowing gas across top surface 31 in substantially themanner as discussed above with regard to nozzle 96. This configurationhas the advantage that lip seal 99 can seal against tube 126 to preventany fluid from passing into port 46.

As depicted in FIG. 5, as top surface 31 of culture 29 rises withincontainer 18 to a location at or above passageway 94 of port 46, tube126 can be advanced further into container 28 by advancing second end122 of sleeve 118. As a result of the resilient arch of tube 126, tube126 naturally curves upward within chamber 28 so that nozzle 132 canremain positioned above top surface 31 of culture 29 for blowing gasacross top surface 31. As such, delivery system 110 can operate toproduce gas stream oxygenation over a range of different elevationallevels of top surface 31, i.e., from below passageway 94 to abovepassageway 94. The distances, dimensions, velocities, flow rates,orientations and the like discussed above with regard to gas deliverysystem 60A and passageway 66 are also applicable to gas delivery system110 and outlet 134.

Depicted in FIG. 6, is yet another alternative embodiment of a gasdelivery system 140. Like elements between gas delivery systems 110 and140 are identified by like characters. Gas delivery systems 110 and 140are substantially the same except that in system 140 an upwardly curveddirecting stem 142 is coupled with stem 92 of port 46. Directing stem142 can be integrally formed with port 46 or can be separately attachedthereto by having a portion of directing stem 142 be received within andclamped onto stem 92. Directing stem 142 has a passageway 144 extendingtherethrough that communicate with passageway 94. In this configuration,as sleeve 118 is collapsed, first end 120 of tube 126 advances throughport 46 and into passageway 144 of directing stem 142. As a result ofthe curvature of directing stem 142, first end 120 is then curvedupwards where it exists into compartment 28. As a result of thecurvature of directing stem 142, tube 126 need not have a resilientcurvature but can comprise a standard flexible tube.

Tube 126 has a nozzle 146 mounted on the end thereof. Formed on the sideof nozzle 146 are a plurality of radially spaced apart outlets 148through which the gas stream outwardly flows. By adjusting the verticalposition of nozzle 146, delivery system 140 can again operate over arange of elevations of top surface 31 of culture 29.

FIG. 7 depicts another alternative embodiment of a gas delivery system163 for delivering a gas stream over top surface 31 of culture 29 forachieving gas stream oxygenation/mass transfer. Gas delivery system 163can be used independent of or in combination with the other gas deliverysystems discussed herein. Gas delivery system 163 comprises a tubularsleeve 162 having a first end 164 and an opposing second end 166. Sleeve162 has an interior surface 168 that bounds a passageway 170 extendingalong the length thereof. Sleeve 162 is collapsible and can be made ofthe same materials and have the same alternative configurations andproperties as previously discussed with regard to sleeve 118 (FIG. 6).

Secured to upper end wall 33 of container 18 is a tubular port 171having a tubular stem 172 projecting therefrom into compartment 28.Second end 166 of sleeve 162 is coupled in sealed engagement to tubularstem 172. Mounted on first end 164 of sleeve 162 is a nozzle 174. Nozzle174 comprises a body 176 having a first end face 178 and an opposingsecond end face 180 and an encircling sidewall 182 extendingtherebetween. A plurality of outlets 184 are formed on sidewall 182 atradially spaced apart locations around sidewall 182. Outlets 184 canalso be formed on first end face 178. Outwardly projecting from secondend face 184 of nozzle 174 is a tubular stem 186 coupled in sealedengagement with first end 164 of sleeve 162.

Gas delivery system 163 further comprises a tube 189 having a first endthat extends down through passageway 170 of sleeve 162 and fluid coupleswith nozzle 174 and an opposing second end that is disposed outside ofcontainer 18 and couples with a gas supply 82A. Although tube 189 cancomprise a single continuous tube, in the depicted embodiment tube 189comprises a first tube portion 190 and a second tube portion 196. Firsttube portion 190 is disposed within passageway 170 of sleeve 162 and hasa first end that is fluid coupled with nozzle 174 so that gas travelingdown through first tube portion 190 passes out through outlets 184. Afirst connector portion 192 is disposed at an opposing second end offirst tube portion 190. A gas filter 84 is disposed along first tubeportion 190 so that the gas passing therethrough is sterilized.

Second tube portion 196 has a first end with a second connector portion193 mounted thereon. Connector portions 192 and 193 can be selectivelycoupled together to form a fluid tight connection therebetween.Connector portions 192 and 193 typically form a sterile connector suchas previously discussed connector 72 (FIG. 2). Second tube portion 196passes out of sleeve 162 and container 18 by passing through tubularport 172. An opposing second end of second tube portion 196 is coupledwith gas supply 82A. Thus, in the assembled configuration, gas from gassupply 82A can travel through second tube portion 196, first tubeportion 190, and out through outlets 184 of nozzle 174 so as to flowover top surface 31 of culture 29.

Second tube portion 196 can be coiled around a spool. As top surface 31of culture 29 rises within container 18, the spool can be rotated sothat more of tube 189 is wound around the spool. In so doing, nozzle 174is lifted so that outlets 184 are always maintained at a desiredelevation above top surface 31. As nozzle 174 is lifted, sleeve 162simply collapses or compresses. In contrast, as top surface 31 lowers,tube 189 is unwound from the spool causing nozzle 174 to lower andsleeve 162 to expand. The distances, dimensions, velocities, flow rates,orientations and the like discussed above with regard to the othernozzle outlets are also applicable to outlets 184. It is likewiseappreciated that a spool is not required for tube 189 and that any typeof lift can be used to raise and lower tube 189. In yet anotherembodiment, a line, such as a rope or cable, can be passed down sleeve162 and coupled with nozzle 174 for raising and lowering nozzle 174 sothat no undue stress is applied on tube 189 and connector 192/193.Sensors can be used to detect the height of top surface 31 andautomatically adjust the height of nozzle 174 accordingly.

In another embodiment, nozzle 174 can be configured to float. This canbe accomplished by making nozzle 174 out of a buoyant material or bysecuring a float to nozzle 174. As a result, nozzle 174 can restdirectly on top surface 31 of culture 29 and then automatically raiseand lower as top surface 31 raises and lowers. A spool or other lift canstill be used for gathering and releasing tube 189.

Gas delivery system 163 is configured so that first tube portion 190,sleeve 162 and nozzle 174 can be preassembled with and sterilizedconcurrently with container 18. During use, first connector portion 192can be slid out of sleeve 162 through port 171 and connected with secondconnector portion 193. After use, connector portions 192/193 can bedisconnected and the container assembly disposed of.

Gas delivery system 163 achieves the same function of producing gasstream oxygenation/mass transfer with culture 29 as the previouslydiscussed gas delivery systems. However, gas delivery system 163 has thefurther advantage that nozzle 174 can be more centrally located on orabove top surface 31 and can dispense gas radially outwardly so as tomore uniformly apply the gas over all or most of the area of top surface31. Furthermore, the mass transfer can be more constantly maintainedbecause outlets 184 can be continuously maintained at a desiredelevation above top surface 31.

Depicted in FIG. 8 is yet another alternative embodiment of a gasdelivery system 200 that incorporates features of the present inventionand can achieve gas stream oxygenation/mass transfer with culture 29.Gas delivery system 200 can also be used in conjunction with orindependent of the other gas delivery systems disclosed herein. Gasdelivery system 200 comprises a tube 210 having a first end 212 and anopposing second end 214. As depicted in FIG. 9, tube 210 has a pluralityof lumens 215 extending along at least a portion of the length of tube210. A plurality of nozzles 216A-G are coupled with tube 210 at spacedapart location along the length thereof. Each nozzle 216 encircles andradially outwardly projects from tube 210. Each nozzle 216 has a topsurface 217, an opposing bottom surface 219 and an encircling sidesurface 218 extending therebetween. A plurality of outlets 220 areformed of side surface 218 at radially spaced apart locations aroundnozzle 216. Outlets 220 can also be formed on bottom surface 219. Thedistances, dimensions, velocities, flow rates, orientations and the likediscussed above with regard to the other nozzle outlets are alsoapplicable to outlets 220.

Each lumen 215 of tube 210 has an opening that communicates with acorresponding nozzle 216A-G. For example, as depicted in FIG. 10, withinnozzle 216A an opening 222 is formed on tube 210 so as to communicatewith a lumen 215A. As such, gas traveling down through lumen 215A passesout of opening 222 and into nozzle 216A. The gas radially passes out ofnozzle 216A through outlets 220 so that it can pass over top surface 31of culture 29. Each nozzle 216B-G is similarly configured with acorresponding opening 222 communicating with a separate lumen 215therein.

Returning to FIG. 8, second end 214 of tube 210 passes in sealedconnection through port 40 so as to be disposed outside of container 18.A separate secondary tube 224 is fluid coupled with each lumen 215. Theopposing end of each secondary tube 224 is fluid coupled with a manifold226 which in turn is fluid coupled to a gas supply 228. A separate valve230 is disposed along each secondary tube 224 and is controlled by acentral controller. Accordingly, as culture 29 raises and lowers withincontainer 18, the controller selectively opens or closes valves 230 sothat only the nozzle 216 that is directly above top surface 31 has gasflowing therethrough. For example, in FIG. 8, only the valve 230 coupledto the secondary tube 224 that feeds gas to nozzle 216F would be opened.The remaining valves 230 would be closed. For nozzles 216 that are belowtop surface 31 and thus within culture 29, it is appreciated thatcorresponding valves 230 can be slightly opened so that a positive gaspressure is produced within the submerged nozzle 216 and therebypreclude fluid from entering therein. If desired, a positive pressurecan also be applied to the other nozzles 216 that are not in use.

As top surface 31 of culture 29 raises to nozzle 216F, gas would beclosed off to nozzle 216F and opened to nozzle 216E. Again, gas passingout through nozzles 216 flows over top surface 31 so as to produce a gasstream oxygenation/mass transfer therewith. As with gas delivery system163, gas delivery system 200 has the advantage that nozzles 216 can bemore centrally located on or above top surface 31 and can dispense gasradially outwardly so as to more uniformly apply the gas over all ormost of the area of top surface 31. Furthermore, multiple nozzles 216can be easily placed at adjacent locations along tube 210 so that thegas stream is more uniformly maintained on top surface 31, thereby moreconsistently maintaining the mass transfer.

In addition to the above, it is appreciated that there are other gasdelivery systems that can be used to deliver a gas stream into container28 to achieve gas stream oxygenation/mass transfer. In addition, it isappreciated that the gas need not be delivered through side 20 ofcontainer 20. For example, by adjusting the size of the passagewayextending through access port 40 (shown in FIG. 1) or by positioningnozzle 96 (shown in FIG. 3) therein, a gas flow could be passedtherethrough at a sufficient flow rate and speed so that the gas willtravel downwardly from port 40 to the top surface 31 and then will bedeflected to travel horizontally or substantially horizontally acrosstop surface 31 so as to produce gas stream oxygenation of culture 29. Inother embodiments, gas delivery system 140 shown in FIG. 6, minusdirecting stem 142, could be mounted on upper end wall 33 (shown in FIG.1). In this embodiment, tube 126 could be lowered within compartment 28until nozzle 146 is located a proper distance above top surface 31 toproduce gas stream oxygenation/mass transfer. In turn, as the level ofculture 29 increases within compartment 28, tube 126 and nozzle 146 canbe drawn upward so that nozzle 146 remains at the optimal position forgas stream oxygenation/mass transfer. This embodiment is similar to gasdelivery system 163.

Returning to FIG. 1, reactor system 10 also includes mixing system 17which is used to mix culture 29 so that it remains substantiallyhomogeneous and is uniformly oxygenated. In the depicted embodiment,mixing system comprises a drive shaft 262 that projects into container18 and an impeller 260 mounted on drive shaft 262 so that as drive shaft262 rotates, impeller 260 rotates within culture 29. A dynamic seal 264forms an aseptic seal between drive shaft 262 and container 18 whilestill enabling rotation of drive shaft 262. In some embodiments, theterm “impeller” can refer to any device that is used for agitating ormixing the contents of a stirred-tank reactor system (e.g., bioreactor).The impeller may agitate the fluidic medium by stirring or othermechanical motion. Examples of impellers that can be used in the presentinstant invention include, but are not limited to, a Rushton, a marine,a hydrofoil, a pitched blade, and any other commercially availableimpeller.

It is appreciated that a variety of other mixing systems can also beincorporated into reactor system 10. For example, in contrast to driveshaft 162 rotating impeller 160, a drive shaft can be used thatvertically raises and lowers an impeller or mixing element for mixingthe culture. In yet other embodiments, the drive shaft can be eliminatedand impeller 160 can be magnetically driven. In still other embodiments,the drive shaft and impeller can be eliminated and mixing can beaccomplished by rocking container 18 so that the culture is mixedtherein. In this embodiment, container 18 can be placed on a rockertable rather than being disposed within support housing 12. A pulsatingdisk, paddle mixer, or sill other mixing elements can also be used formixing the culture. Other embodiments for driving an impeller within areactor are disclosed in US Publication No. 2013/0101982, published Apr.25, 2013, which is incorporated herein by specific reference in itsentirety. The above examples for mixing culture 29 are examples of meansfor mixing culture 29 or other fluid that may be contained withincontainer 12.

The above methods and systems have primarily been discussed inassociation with oxygenating a culture within a reactor. As previouslymentioned, however, that the same methods and systems for passing a gasstream over a fluid surface to achieve gas stream mass transfer can alsobe used for other purposes. For example, the inventive methods andsystems can be used for entraining oxygen or other types of gases intofluids other than a culture. In other embodiments, in contrast tooxygenating through a gas stream that passes over the top surface of theculture, oxygenation in a reactor could be accomplished by conventionalmechanisms through a sparger, such as sparger 52. The inventive systemcould then be used to deliver a stream of pure nitrogen over the surfaceof the culture. The nitrogen would be transferred into the culture andused to strip CO₂ from the culture. The same process can also be usedfor stripping other gases from other types of fluids. Thus, theinventive gas stream mass transfer can be used for both entraining a gasinto a fluid and stripping a gas from a fluid. The circulation producedby the nitrogen stream or other inert gasses could also entrain oxygenintroduced through inlet port 40 to supplement oxygen provided by thesparger.

In another example, when it is desired to operate an anaerobic system,nitrogen or other inert gasses could be passed over the surface of theculture so as to purge out oxygen from the culture. Accordingly, theinventive methods and systems disclosed herein can be used in anybiological, chemical, food or other processing or production where it isdesired to affect a mass transfer of any gas into or out of a liquid.

The present invention may be embodied in other specific forms withoutdeparting from its spirit or essential characteristics. The describedembodiments are to be considered in all respects only as illustrativeand not restrictive. The scope of the invention is, therefore, indicatedby the appended claims rather than by the foregoing description. Allchanges which come within the meaning and range of equivalency of theclaims are to be embraced within their scope.

What is claimed is:
 1. A method for performing a gas-liquid masstransfer, the method comprising: (a) mixing a liquid within acompartment of a container, the liquid having an exposed top surfacedisposed within the compartment of the container; (b) moving a tubewithin the container so that an outlet on the tube or on a nozzleconnected to the tube is positioned within a head space at a locationabove the top surface of the liquid; and (c) blowing a stream of gasinto the head space through the outlet so that the stream of gas isblown over at least a portion of the top surface of the liquid while theliquid is being mixed within the container so as to produce a gas-liquidmass transfer between the gas and the liquid.
 2. The method as recitedin claim 1, wherein the liquid comprises a biological suspensioncomprised of cells or microorganisms suspended within a growth medium.3. The method as recited in claim 1, wherein the container comprises aflexible bag having a bottom wall, a top wall and an encircling sidewallextending therebetween, the step of blowing the stream of gas comprisingdelivering the gas through a first opening formed on the container. 4.The method as recited in claim 1, wherein the step of blowing the streamof gas comprises delivering the gas through the nozzle coupled to ordisposed within the compartment of the container.
 5. The method asrecited in claim 1, wherein the stream of gas produces a kLa factor inthe liquid of at least
 3. 6. The method as recited in claim 1, whereinthe step of moving the tube comprises: advancing the tube through afirst opening formed on the container so that a first end of the tube islocated within the compartment of the container.
 7. The method asrecited in claim 1, wherein the step of blowing the stream of gas overthe at least a portion of top surface of the liquid occurs withoutsparging a gas into the liquid.
 8. The method as recited in claim 1,wherein the top surface of the liquid is at a first elevation within thecontainer during the step of blowing the stream of gas, the step ofblowing the stream of gas over the at least a portion of the top surfaceof the liquid occurring without sparging a gas into the liquid while theliquid is at the first elevation, and the method further comprising: a)adding a growth medium to the liquid within the container so as to raisethe top surface of the liquid to a second elevation within thecompartment; and b) sparging a gas into the liquid after the top surfacehas been raised to the second elevation within the compartment.
 9. Themethod as recited in claim 1, wherein the step of mixing the liquidcomprises rotating a mixing element within the compartment of thecontainer.
 10. The method as recited in claim 1, wherein the step ofmixing is performed independent of blowing the stream of gas.
 11. Themethod as recited in claim 1, wherein the step of moving the tubecomprises moving at least a portion of the tube vertically within thecompartment of the container.
 12. A method for performing a gas-liquidmass transfer, the method comprising: (a) mixing a liquid within acompartment of a container, the liquid having an exposed top surfacedisposed within the compartment of the container; (b) advancing a tubewithin a first opening formed on the container so that a first end ofthe tube is positioned at a desired location above the top surface ofthe liquid within the compartment of the container; and (c) blowing astream of gas out the first end of the tube so that the stream of gaspasses over at least a portion of the top surface of the liquid whilethe liquid is being mixed within the container so as to produce agas-liquid mass transfer between the gas and the liquid.
 13. The methodas recited in claim 12, wherein the step of blowing the stream of gasover the at least a portion of top surface of the liquid occurs withoutsparging a gas into the liquid.
 14. The method as recited in claim 12,wherein the step of mixing the liquid comprises rotating a mixingelement within the compartment of the container.
 15. The method asrecited in claim 12, wherein the liquid comprises a biologicalsuspension comprised of cells or microorganisms suspended within agrowth medium.
 16. The method as recited in claim 12, wherein the stepof advancing the tube comprises moving at least a portion of the tubevertically within the compartment of the container.
 17. A method forperforming a gas-liquid mass transfer, the method comprising: (a) mixinga liquid within a compartment of a container, the liquid having anexposed top surface disposed within the compartment of the container;(b) blowing a stream of gas over at least a portion of the top surfaceof the liquid while the liquid is being mixed within the container so asto produce a gas-liquid mass transfer between the gas and the liquid,wherein the top surface of the liquid is at a first elevation within thecontainer during the step of blowing the stream of gas, the step ofblowing the stream of gas over the at least a portion of the top surfaceof the liquid occurring without sparging a gas into the liquid while theliquid is at the first elevation; (c) adding a further liquid to theliquid within the container so as to raise the top surface of the liquidto a second elevation within the compartment; and (d) sparging a gasinto the liquid within the container after the top surface has beenraised to the second elevation within the compartment.
 18. The method asrecited in claim 17, wherein the liquid comprises a biologicalsuspension comprised of cells or microorganisms suspended within agrowth medium.
 19. The method as recited in claim 17, wherein thecontainer comprises a flexible bag.
 20. The method as recited in claim17, wherein the step of mixing the liquid comprises rotating a mixingelement within the compartment of the container.